The long-term goal of our research is to understand transcription mechanisms of cellular RNA polymerase (RNAP) and its regulation. During the next project period, we will study the transcription machinery in bacteria to provide a fundamental mechanism of transcription, which is conserved from bacteria to human. Recently, we reported the first X-ray structure of the Escherichia coli RNAP s70 holoenzyme. This enzyme is the most studied RNAP and has been used as a model RNAP for understanding the mechanism of transcription. E. coli RNAP is conveniently prepared using an overexpression system, which allows exploring new directions in RNAP structural studies including the transcription elongation complex, paused transcription complex, RNAP in complex with a variety of transcription factors and inhibitors, as well as RNAP mutants. Here, we propose structural and biochemical studies of E. coli RNAP transcription to address three specific aims. Aim1. Structural basis for productive-phase transcription: We crystallized an E. coli RNAP elongation complex and determined its X-ray structure at 6 resolution, which provides a framework for the structure- based study of the transcription mechanism. Further experiments are proposed: (1) to determine the atomic resolution structure of the elongation complex for analyzing interactions between RNAP and nucleic acids; (2) to determine structures of the elongation complex with elongation factors NusG or RfaH for determining the positions of the non-template DNA in the transcription bubble and the upstream DNA for the first time in the context of an intact elongation complex; and (3) to determine the structure of the elongation complex with an RNAP mutant prone to transcription slippage for understanding transcriptional fidelity. Our E. coli RNAP elongation complex crystal can extend multiple RNA bases in crystal form. Therefore, we will carry out in crystallo transcription and record motions of the bridge helix and trigger loo as well as translocation of nucleic acids during transcription elongation using time-resolved soak-trigger-freeze X-ray crystallography. Aim 2. Elucidate the molecular mechanism of transcription pausing by the pause-trigger sequence: Nascent transcript sequencing of the E. coli transcriptome identified a consensus pause sequence in the E. coli genome. We will determine the crystal structure of the elongation complex containing the consensus pause sequence to reveal the interplay between RNAP and nucleic acids during transcription pausing and to provide novel insight into gene regulation. Aim 3. Structural basis for RNAP modulation by NusA: Most transcription elongation complexes in vivo associate with NusA, which stimulates the effect of RNA hairpins for pausing and termination. We will determine the crystal structures of NusA in complex with RNAP and also with the elongation complex to elucidate the structural basis for NusA-dependent pausing and termination.